Repair of 8-methoxypsoralen photoinduced cross-links in yeast. Analysis by alkaline step-elution and electron microscopy

Abstract

The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D7 and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced cross-links. In accordance with previous reports, the D7 rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D7 rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D7 RAD+ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D7 rad18-1 than in D7 RAD+. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.